A NOVEL FORMULATION of HERBS with POTENT ANTI-AGEING ACTIVITY Mohit M

European Journal of Bioinformatics, Vol. 2:1-5, 2014

A NOVEL FORMULATION OF HERBS WITH POTENT ANTI-AGEING ACTIVITY Mohit M. Jaina, Nirmala Kumaria, Geeta Raib* aNEISS Wellness India Limited, Mumbai, India. bDepartment of Molecular and Human Genetics, Banaras Hindu University, Varanasi 221005, India.

ABSTRACT Ageing is characterized by a progressive deterioration of physiological functions and metabolic processes. Sirtuin (SIRT1) is a NAD+-dependent protein deacetylase which is a human ortholog of the yeast SIR2 protein. The activation of SIRr2 affects wide variety of cellular processes ranging from metabolism, cell cycle, growth and differentiation, inflammation, senescence, apoptosis, stress response and ageing. There are natural anti- ageing drugs in the market like Resveraterol but some studies suggest that due to its interaction with contraceptive pills it should not be consumed by women taking them. Although, a diet rich in antioxidants seems to offer hope in delaying the onset of unhealthy disorders that accompany ageing, however no clinical treatment using natural herbs has yet been developed and natural anti-aging drugs are still unavailable. In the present study, we evaluated the binding of active ligands present in real grass with the SIRT1 and SIRT5 receptor and compared their binding energy and affinity with other reference anti-ageing drugs present in the market. A high throughput screening comprising of molecular docking, absorption, distribution, metabolism, excretion and toxicity predictions, logP values and percentage of human oral absorption value led to the identification of three potential compounds present in ‘Live Green Real Grass’ which could be considered for anti–ageing activity with better binding affinities than the reference drugs used. KEYWORDS : Anti-ageing, Reservetrol, SIRT, real grass ABBREVIATIONS : SIRT 1 / SIRT 5 - silent mating type information regulation 2 homolog 1 /5 FOXO - Forkhead-O-box NAD+ - Nicotinamide adenine dinucleotide

INTRODUCTION towards healthy ageing, by modifying their lifestyle and adopting ways for prevention of diseases by In recent years owing to modernization, life healthy diet. style related disorders are on the rise at an exponential rate. Natural ageing depends greatly Many theories and mechanisms have been on diet and lifestyle. It is a fact that for those with proposed for premature ageing that can be healthier body aging is delayed compared to those targeted to slow down the ageing process. Five who lead a life of frequent smoking, alcohol major theories of ageing are: consumption, eating junk food and a sedentary i) Antioxidant/free radical theory, ii) lifestyle (Guarente et al., 2009). Lifestyle effects are Mitochondrial mutation theory, iii) Cell debris/ controlled by proteins called sirtuins, which keep cleansing theory, iv) Hormonal changes, and v) cells live and healthy, even in stress, by regulating Calorie restriction theory. According to the free many hormones and their networks, regulatory radical theory (Harman, 1992), free radicals proteins and other genes (Guarente et al., 2009). produced during metabolic processes cause Today, several health issues resulting from poor degeneration of cells contributing towards diet and lifestyle as well as early symptoms of ageing. ageing are seen in people as early as in their 20’s. According to McLean et al., 2004, the primary Mitochondrial theory of aging states that causes of early ageing are stress, exposure to ageing in humans and animals is primarily due to harmful chemicals, pollutants, etc. Due to the accumulation of damages caused to the increased awareness people are now shifting mitochondria and mitochondrial DNA (Wei et al.,

European Journal of Bioinformatics, Vol. 2:1-5, 2014

2001). stress, enhances the energy level thereby reduces premature ageing. Cuervo, 2008 in her cleansing theory states that as we age, lysosomes increasingly fail to perform Schuetz et al., 2007 states that SIRT5 is a NAD+ cleansing of the cells, leading to cell death and this dependent deacetylase, localized in mitochondrial leads to premature ageing.Vanja et al., 2010 in his matrix and activation of it is known to deacetylate hormonal theory states that the changes that the mitochondrial enzyme carbomoyl phosphate occur in the body because of the hormonal system synthetase 1 (CPSl), cytochrome C and a protein of are one of the very crucial reasons of ageing. As the mitochondrial inter-membrane space with a per calorie restriction theory proposed by McCay central function in oxidative metabolism (Schlicker et al., (1989), one should reduce the calorie et al., 2008). Therefore, these molecules can be consumption to increase in longevity. Sirtuin targeted to slow down the process of early or enzymes form a closed family of Nictotinamide premature ageing.The current study aims to adenine dinucleotide (NAD)-dependent examine the phytocompounds from a dietary deacetylases and ADP-ribosyltransferases, which is supplement, ‘Live Green Real Grass’ with anti- a life-span regulator in lower organisms. Chen et ageing and anti-oxidant properties for their al., (2005) and Haigis et al., (2010) state that there efficiency against anti-ageing targets (SIRT1 and are several genes in the sirtuin class, which SIRT5).The list of the phyto-nutrients identified is includes SIRT1, SIRT3, and SIRT4 that produce included in Table 1. enzymes which increase the activity of the mitochondria and slow down the cell's process of MATERIALS & METHODS ageing. As stated by Li et al., (2008) and Nakagawa 2.1 Active structure retrieval et al., (2009) Sirtuins proteins (SIRT1-7) regulate biological pathways, including energy metabolism The structures of active compounds of the (Li et al., 2008 and Nakagawa et al., 2009). botanicals used in the composition of the product Live Green Real Grass were acquired from As mitochondria is a key player in metabolism, PubChem(http://pubchem.ncbi.nlm.nih.gov) energy maintenance, and apoptosis, disruption of (Figure 1). mitochondrial pathways can lead to metabolic disease, oxidative damage, and cancer. Restoration 2.2 Preparation of receptor of these mitochondrial pathways may help in SIRT1 protein structure was downloaded from treating or preventing metabolic diseases, Protein Data Bank (PDB) with PDB ID - 4FZ3. The oxidative damage, and cancer. Therefore, Sirt1 and resolution factor of this structure was 2.10 Å and Sirt5 receptor present in mitochondria can be the method of incorporation was X-ray diffraction. targeted to slow down the process of early or PDB file of NAD-dependent protein deacylase premature ageing. sirtuin-1, receptor (PDB ID: 4FZA) has 2 chains. Sirtuins proteins (SIRT1-7) regulate various Chain A (NAD-dependent protein deacetylase biological pathways that are directly linked to anti– sirtuin-3, mitochondrial) contains 283 amino acid ageing such as ATP generation, metabolism, calorie residues and Chain B (Fluor-de-Lys peptide) restriction, autophagy and apoptosis. As stated by contains 6 amino acid residues. The proteins were Hirschey et al., (2010) SIRT1, SIRT6, and SIRT7 are preprocessed separately by deleting the substrate nuclear proteins, SIRT2 is found primarily in cofactor as well as the crystallographically cytosol, and SIRT3, SIRT4, and SIRT5 are observed water molecules (water without H mitochondrial proteins (Hirschey et al., 2010). bonds), correcting the mistakes in PDB file and SIRT1 interact with FOXO family of transcription optimizing hydrogen bonds. factors thereby deacetylates these transcription The protein SIRT5 was downloaded from factors (Brunet et al., 2004; Motta et al., 2004). Protein Data Bank with PDB ID - 4HD with a Further it regulates various processes like lipid resolution factor of 2.60 Å and the method of metabolism, stress resistance, and apoptosis (Gross incorporation being, X-ray diffraction. PDB file of et al., 2008) and enhances autophagy (Hariharan NAD-dependent protein deacylase sirtuin-5, et al., 2010). It was observed by Brunet et al., mitochondrial receptor (PDB ID: 3HDA) has 2 (2004) Deacetylation of FOXO3 by SIRT1 not only chains. Chain A (NAD-dependent protein deacylase inhibits its activity on apoptosis-related gene sirtuin-5, mitochondrial) contains 275 amino acid expression but drives its actions toward the residues and Chain F (Fluor-de-Lys peptide) induction of oxidative stress resistance genes contains 4 amino acid residues. The proteins were (Brunet et al., 2004). This is in line with the preprocessed separately by deleting the substrate hypothesis that activation of SIRT1 may allow the cofactor as well as the crystallographically cell to manage with high stress level/oxidative 2

European Journal of Bioinformatics, Vol. 2:1-5, 2014 observed water molecules (water without H We used resveratrol as our reference drug bonds), correcting the mistakes in PDB file, and which is a small polyphenolic compound which act optimizing hydrogen bonds. as potent natural anti-ageing agent. De la Lastra et al., 2005 reported resveratrol to be an activator of

2.3 Compounds used for study the sirtuin family of protein deacetylases. In this In this study we used the active compounds of study we have evaluated the mode of interaction the cereal grasses used in the composition of the of active ingredients of Live Green Real Grass with product Real Grass. The main active ingredients in the SIRT receptors. We used two docking methods, Real Grass are Abcissic acid, Chlorophyll, Moldock and Hex for evaluation of interaction of Spinasterol, stigmasterol, Beta-sitosterol and receptors and individual active compounds present Catechin-7-o-glucoside. The 2D structures of all in Live Green Real Grass. The comparative studies these active ingredients present in ‘Live Green Real of two docking results are shown in Table 2. Grass’are shown in Figure 2. Among all the natural activators docked within the active site of the SIRT1 and SIRT5 receptor, 2.4. Ligands preparation chlorophyll, beta-sitosterol and stigmasterol were The structure of the active compound of the found to bind with high affinity within the active vegetables used in the composition of the product site pocket of SIRT5 receptor as well as with SIRT1 Real Grass were retrieved from receptor; spinasterol and catechin7 o glucoside PubChem(http://pubchem.ncbi.nlm.nih.gov). were also effective. All the comparisons were made Further the explicit hydrogen bonds were added to on the basis of docking scores obtained from the active structure and then 3D conformers were Moldock and Hex software. These three generated via Marvin Sketch. Conformer with ingredients showed better docking than reference lowest energy was used for docking studies. drug. Of all the ingredients chlorophyll, beta- sitosterol and stigmasterol formed seven, four and 2.5. Docking methods three hydrogen bonds with SIRT5 receptor, We used two docking softwares namely Hex respectively (as shown in Figure 2d, 2e and 2f). and MolDockfor studying the interactions of all Interaction diagram (2 dimensional) of spinasterol these active ingredients with SIRT 1 and SIRT 5 and catechin 7 o glucoside with SIRT5 receptor also receptor. The basic aim of docking procedures was depicts five and seven H bond interactions, to identify the correct conformation of ligands in respectively (Figure 2e and 2b). Ligand interaction the binding pocket of a protein and to predict the studies with SIRT1 receptor showed that affinity between the ligands and the receptor. All chlorophyll, beta–sitosterol and stigmasterol the docking results were evaluated on the basis of formed nine, seven and four hydrogen bonds with docking score as well as the interaction study with SIRT1 Receptor, respectively (as shown in Figure the active side residues of the SIRT 1 and SIRT 5 3d, 3e and 3f). Interaction diagram (2 D) of receptor. spinasterol and catechin7 o glucoside with SIRT 1 receptor also depicts nine and six hydrogen bond 2.6. 2 D diagram preparation interactions, respectively (Figures 3e and 3b). For studying hydrogen bonds as well as the In silico pharmacokinetics study was done with hydrophobic interactions of all docking results 2D ACD Lab tools for all the six ingredients present in diagrams were drawn. The 2D structure showing the Live Green Real Grass and they were evaluated the hydrogen bonds, steric interaction as well as for their levels of absorption. Chlorophyll, Beta– electrostatic interactions were produced by ligand sitosterol and stigmasterol showed the highest oral interaction tool of MolDock docking tool. passive absorption (100%) (Table 3) In addition to its oral absorption capacity, it has also showed the 2.7. ADME/T properties prediction: capacity of crossing the blood brain barrier; thus Absorption, distribution, metabolism, excretion can act directly on central nervous system and toxicity (ADME/T) properties of all the active activating SIRT1 and SIRT5 receptor signaling. Of all ingredients were predicted using ACD Lab tool the ingredients, phytosterols showed sufficient (ACD/ADME- Tox, version 12.01). It predicts probability of crossing the blood brain barrier. physically significant descriptors and Whereas resveraterol on other side too crosses the pharmaceutically relevant physiochemical blood brain barrier but may stimulate the growth properties of the active ingredients present in ‘Live of human breast cancer cells, because of its Green Real Grass’ like LogP, pKa, pKb as well as chemical structure, which is similar to a their ability to cross the blood brain barrier. phytoestrogen (Gehm et al., 1997; Bowers et al., 2000). It may interfere with oral contraceptives and RESULTS AND DISCUSSION has adverse effect on woman who is pregnant or 3

European Journal of Bioinformatics, Vol. 2:1-5, 2014 intending to become pregnant. 8) McCay, C.M., Crowell, M.F., Maynard, L.A, 1935. The effect of retarded growth upon the CONCLUSION length of life span and upon the ultimate body These in silico molecular docking study and size.Journal of Nutrition, 10:63-79. pharmacokinetics study reveal that Real Grass may act as a probable dietary supplement for anti- 9) Weindruch, R., Walford, R.L, 1982. Dietary ageing. In the present study, we report that the restriction in mice beginning at 1 year of age: active ingredients present in Real Grass, namely effect on life-span and spontaneous cancer chlorophyll ,beta–sitosterol and stigmasterol, are incidence. Science, 215(4538): 1415-1418. the potential agonist of SIRT1 and SIRT5 receptor 10) Roy, W., M.D, 2000. Beyond the 120 Year Diet: present in the mitochondria of the cells in different How to Double Your Vital Years'. organs like brain. As stated by Haigis et al., 2006 that mitochondria are the center stage for cellular 11) Chen, D., Steele, A.D., Lindquist, S., Guarente, energy (ATP) production, generation of ROS, and L, 2005. Increase in activity during calorie signaling during apoptosis thereby helps in restriction requires Sirt1. Science,310:1641. enhancing longevity, cell-cycle regulation, gene silencing, stress response, insulin sensitivity, 12) Haigis, M.C., Mostoslavsky, R., Haigis, K.M., gluconeogenesis, lipid metabolism, axonal Fahie, K., Christodoulou, D.C., Murphy, A.J., degeneration and apoptosis . Valenzuela, D.M., Yancopoulos, G.D., Karow, We propose that the patients consuming live Blander, M.G., Wolberger, C., Prolla, T.A., green Real Grass may achieve longevity at a faster Weindruch, R., Alt, F.W., Guarente, L, 2006. rate compared to resveratrol. IRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in REFERENCES pancreatic beta cells. Cell, 126: 941-954.

1) McLean, A.J., Le Couteur, D.G, 2004. Aging 13) Hirschey, M.D., Shimazu, T., Goetzman, E., Biology and Geriatric Clinical Pharmacology. Jing, E., Schwer, B., Lombard, D.B., Grueter, Pharmacological Reviews, 56(2): 163-184. C.A., Harris, C., Biddinger, S., Ilkayeva, O.R., Stevens, R.D., Li, Y., Saha, A.K., Ruderman, 2) Kulkarni, S.D., Tilak, J.C., Acharya, R., Rajurkar N.B., Bain, J.R., Newgard, C.B., Farese, R.V., N.S., Devasagayam, T.P.A., Reddy, A.V.R, 2006. Alt, F.W., Kahn, C.R., Verdin E, 2010. SIRT3 Evaluation of the antioxidant activity of regulates mitochondrial fatty-acid oxidation by wheatgrass (Triticumaestivum L.) as a function reversible enzyme deacetylation. Nature , of growth under different 464:121-125. conditions.Phytotherapy Research, 20(3): 218- 227. 14) Gross, D.N., Van den Heuvel, A.P., Birnbaum, M.J, 2008. The role of FoxO in the regulation 3) Harman, D, 1992. Free radical theory of of metabolism.Oncogene.27(16): 2320–2336. aging.Mutat Res, 275(3-6):257-66. 15) Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, 4) Wei, Y.H., Ma, Y.S., Lee, H.C., Lee, C.F., Lu, C.Y, K.F., Greer, P.L., Lin, Y., Tran, H., Ross, S.E., 2001. Mitochondrial theory of aging matures- Mostoslavsky, R., Cohen, H.Y., Hu, L.S., Cheng, roles of mtDNA mutation and oxidative stress H.L., Jedrychowski, M.P., Gygi, S.P., Sinclair, in human aging.Zhonghua Yi XueZaZhi Volume, D.A., Alt, F.W., Greenberg, M.E, 2004. Stress- 64(5): 259-70. dependent regulation of FOXO transcription factors by the SIRT1 deacetylase.Science , 5) Cuervo, A. M, 2008. Autophagy and aging. 303(5666) : 2011-2015. Trends Genet, 24(12): 604–612. 16) Motta MC, Divecha N, Lemieux M, Kamel C, 6) Zjacic-RotkvicV.,Kavur, L. and Cigrovski- Chen D, Gu W, Bultsma Y, McBurney M, Berkovic, M, 2010. Hormones and Guarente L, 2004. Mammalian SIRT1 Aging.Diabetes and Metabolism.ActaClin represses forkhead transcription factors. Cell, Croat, 49(4). 116(4): 551–63. 7) McCay, C.M., Crowell, M.F., Maynard, L.A, 17) Hariharan, N., Maejima, Y., Nakae, J., Paik J, 1989. The effect of retarded growth upon the Depinho RA, Sadoshima J, 2010. Deacetylation length of life span and upon the ultimate body of FoxO by Sirt1 Plays an Essential Role in size. Nutrition, 5(3):155-71; discussion 172. Mediating Starvation-Induced Autophagy in 4

European Journal of Bioinformatics, Vol. 2:1-5, 2014

Cardiac Myocytes. Circ Res.,107(12):1470–82. 23) Gehm, B.D., McAndrews, J.M., Chien, P.Y., Jameson, J.L, 1997. Resveratrol, a polyphenolic 18) Li, Y., Xu, W., McBurney, M.W., Longo, V.D, compound found in grapes and wine, is an 2008. SirT1 inhibition reduces IGF-I/IRS- agonist for the estrogen receptor. Proc. Natl. 2/Ras/ERK1/2 signaling and protects neurons. Acad. Sci, 94 (25): 14138–43. Cell Metabolism , 8: 38-48. 24) Bowers, J.L., Tyulmenkov, V.V., Jernigan, S.C., 19) Haigis, M.C., Mostoslavsky, R, Haigis K.M., Klinge, C.M, 2000. Resveratrol acts as a mixed Fahie, K., Christodoulou, D.C. et al, 2006. SIRT4 agonist/antagonist for estrogen receptors inhibits glutamate dehydrogenase and alpha and beta. Endocrinology, 141 (10): opposes the effects of calorie restriction in 3657–67. pancreatic β cells. Cell , 126 : 941–954. 25) Schlicker, C., Gertz, M., Papatheodorou, P., 20) Nakagawa, T., Lomb, D.J., Haigis, M.C., Kachholz, B., Becker, C.F., Steegborn, C. 2008. Guarente, L, 2009. SIRT5 Substrates and regulation mechanisms for the Deacetylatescarbamoyl phosphate synthetase human mitochondrial sirtuins Sirt3 and Sirt5. J 1 and regulates the urea cycle. Cell, 137: 560- Mol Biology, 10;382(3): 790-801. 570. 26) de la Lastra C.A., Villegas I, 2005. Resveratrol 21) Haigis, M.C., Sinclair, D.A, 2010. Mammalian as an anti-inflammatory and anti-aging agent: sirtuins: biological insights and disease mechanisms and clinical implications. relevance. Annu Rev Pathol.5: 253–295. MolNutr Food Res., 49 (5): 405-30. 22) Schuetz, 2007, Cell, 15: 388-389.